Population patch clamp electrophysiology: a breakthrough technology for ion channel screening

Dale, Tim; Townsend, Claire; Hollands, Emma; Trezise, Derek J.

doi:10.1039/b706152h

Cited by 54 publications

(28 citation statements)

References 30 publications

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“…The PPC configuration significantly reduced the heterogeneity in well-to-well signal amplitude (coefficient of variation [CV] <10%), as predicted from the theory of averaging and from experiences with other channels. 23,25,27 This is enabling because it allows meaningful and direct well-to-well signal comparisons. Current voltage-relationships for the isolated GABA response were largely as expected for a gated Cl -conductance under the ionic conditions; I GABA reversed close to the theoretical Cl -equilibrium potential (-48 mV vs. -56 mV) and showed limited rectification.…”

Section: Discussionmentioning

confidence: 99%

Population Patch-Clamp Electrophysiology Analysis of Recombinant GABAA α1β3γ2 Channels Expressed in HEK-293 Cells

Hollands¹,

Dale²,

Baxter³

et al. 2009

SLAS Discovery

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γ-Amino butyric acid (GABA)—activated Cl— channels are critical mediators of inhibitory postsynaptic potentials in the CNS. To date, rational design efforts to identify potent and selective GABAA subtype ligands have been hampered by the absence of suitable high-throughput screening approaches. The authors describe 384-well population patch-clamp (PPC) planar array electrophysiology methods for the study of GABAA receptor pharmacology. In HEK293 cells stably expressing human α1β3γ2 GABAA channels, GABA evoked outward currents at 0 mV of 1.05 ± 0.08 nA, measured 8 s post GABA addition. The IGABA was linear and reversed close to the theoretical ECl (—56 mV). Concentration-response curve analysis yielded a mean pEC50 value of 5.4 and Hill slope of 1.5, and for a series of agonists, the rank order of potency was muscimol > GABA > isoguvacine. A range of known positive modulators, including diazepam and pentobarbital, produced concentration-dependent augmentation of the GABA EC 20 response (1 µM). The competitive antagonists bicuculline and gabazine produced concentration-dependent, parallel, rightward displacement of GABA curves with pA2 and slope values of 5.7 and 1.0 and 6.7 and 1.0, respectively. In contrast, picrotoxin (0.2-150 µM) depressed the maximal GABA response, implying a non-competitive antagonism. Overall, the pharmacology of human α1β3γ2 GABAA determined by PPC was highly similar to that obtained by conventional patch-clamp methods. In small-scale single-shot screens, Z′ values of >0.5 were obtained in agonist, modulator, and antagonist formats with hit rates of 0% to 3%. The authors conclude that despite the inability of the method to resolve the peak agonist responses, PPC can rapidly and usefully quantify pharmacology for the α1β3γ2 GABAA isoform. These data suggest that PPC may be a valuable approach for a focused set and secondary screening of GABAA receptors and other slow ligand-gated ion channels. ( Journal of Biomolecular Screening 2009:769-780)

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Section: Discussionmentioning

confidence: 99%

Population Patch-Clamp Electrophysiology Analysis of Recombinant GABAA α1β3γ2 Channels Expressed in HEK-293 Cells

Hollands¹,

Dale²,

Baxter³

et al. 2009

SLAS Discovery

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“…Accordingly, several methods in vitro are now available to evaluate the potential of drugs to suppress hERG or prolong the QT interval. The high-throughput screening (HTS) system for the hERG inhibition assay, including Rb + flux measurement, 5 potential sensitive fluorescence assay, 6 and improved automated patch clamp system, 7,8 have been also developed. New methods are, however, still emerging to evaluate the potential of test compounds to block the hERG K + channel as early as possible in the drug-development process with lower cost, smaller effort, and no exaggerated equipment.…”

Section: Introductionmentioning

confidence: 99%

Development of Recombinant Cell Line Co-expressing Mutated Nav1.5, Kir2.1, and hERG for the Safety Assay of Drug Candidates

et al. 2012

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To provide a high-throughput screening method for human ether-a-go-go-gene-related gene (hERG) K + channel inhibition, a new recombinant cell line, in which single action potential (AP)-induced cell death was produced by gene transfection. Mutated human cardiac Na + channel Nav1.5 (IFM/Q3), which shows extremely slow inactivation, and wild-type inward rectifier K + channel, Kir2.1, were stably co-expressed in HEK293 cells (IFM/Q3+Kir2.1). In IFM/Q3+Kir2.1, application of single electrical stimulation (ES) elicited a long AP lasting more than 30 s and led cells to die by more than 70%, whereas HEK293 co-transfected with wild-type Nav1.5 and Kir2.1 fully survived. The additional expression of hERG K + channels in IFM/Q3+Kir2.1 shortened the duration of evoked AP and thereby markedly reduced the cell death. The treatment of the cells with hERG channel inhibitors such as nifekalant, E-4031, cisapride, terfenadine, and verapamil, recovered the prolonged AP and dose-dependently facilitated cell death upon ES. The EC 50 values to induce the cell death were 3 µM, 19 nM, 17 nM, 74 nM, and 3 µM, respectively, whereas 10 µM nifedipine did not induce cell death. Results indicate the high utility of this cell system for hERG K + channel safety assay.

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“…The latest automated patch clamp systems make it possible to measure and to average the data from up to 64 cells in a single well immediately (the so-called population patch clamp; Clare 2010). By notably reducing cell-to-cell variability, the population patch clamp provides substantially more precise data (Dale et al 2007).…”

Section: Automated Patch Clampmentioning

confidence: 99%

Advances in patch clamp technique: towards higher quality and quantity

Bébarová¹

2012

gpb

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Abstract. The patch clamp technique, developed in late 1970s, started a new period of experimental cardiac electrophysiology enabling measurement of ionic currents on isolated cardiomyocytes down to the level of single channels. Since that time, the technique has been substantially improved by development of several upgraded modifications providing so far unavailable data (e.g. action potential clamp, dynamic clamp, high-resolution scanning patch clamp), or facilitating the patch clamp technique by increasing its efficiency (planar patch clamp, automated patch clamp). The current review summarizes the leading new patch clamp based techniques used in cardiac cellular electrophysiology, their principles and prominent related papers.

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Population patch clamp electrophysiology: a breakthrough technology for ion channel screening

Cited by 54 publications

References 30 publications

Population Patch-Clamp Electrophysiology Analysis of Recombinant GABAA α1β3γ2 Channels Expressed in HEK-293 Cells

Population Patch-Clamp Electrophysiology Analysis of Recombinant GABAA α1β3γ2 Channels Expressed in HEK-293 Cells

Development of Recombinant Cell Line Co-expressing Mutated Nav1.5, Kir2.1, and hERG for the Safety Assay of Drug Candidates

Advances in patch clamp technique: towards higher quality and quantity

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